J Mol
Cell
Cardiol
22,
1221-1229
(1990)
Net Ca2 + Infhx and Sarcoplasmic Reticulum Ca2 + Uptake in Resting Single Myocytes of the Rat Heart: Comparison with Guinea-pig Bohdan
Lewartowski
and Krzysstof
Zdanowski
Department of Clinical Physiology, Medical Center of Postgraduate Education, Warsaw, Poland (Received 22 January 1990, accepted in revisedform
8 May 1990)
B. LEWARTOWSKI AND K. ZDANOWSKI. Net Ca2+ Influx and Sarcoplasmic Reticulum Ca2+ Uptake in Resting Single Myocytes of the Rat Heart: Comparison with Guinea-pig. Journal of Molecular and Cellular Cardiology (1990) 22, 1221-1229. We tested Shattock and Bers’ (1989) hypothesis according to which in rat cardiac myocytes net Ca2’ influx during diastole via Na/Ca exchange provides the main route of entry of Ca’+ available for activation ofcontractions. We used injections ofcaffeine into the close vicinity of the single, isolated rat or guinea(SR). The cells responded to pig ventricular myocytes in order to release Ca’+ from sarcop lasmic reticulum caffeine with a transient contracture, the amplitude of which was regarded as a relative index of SR Ca2+ content. Application of caffeine deprived the SR of Ca 2+ This was manifested by a very small (rat) or absent (guinea-pig) contractile response to the second application of caffeine and by a decrease of the amplitude of the first post caffeine contraction to 8 + 3% (rat) or to 16 f Sy/, (guinea-pig) of control. In the rat myocytes SR deprived of Ca2+ was able to recover its Ca2+ store even in the resting cell. This was indicated by the time dependent recovery of contractile response to the second application of caffeine and of the amplitude of the postcaffeine electrically evoked contractions. The recovery of post-caffeine contractile responses was completely inhibited by Ca2+ free solution, by 5.0 mM NiC and by low K+ (1 .O mM) hyperpolarising solution superfused from the first application of caffeine or during rest. The recovery was enhanced by superfusion of the cells with low Na+ (50%) solution. These results show that there is a considerable net Ca*+ influx by means of Na/Ca exchange and then the SR Ca*+ uptake in the resting rat myocytes. No recovery of post caffeine contractile responses could be observed in guinea-pig myocytes. KEY
WORDS:
Heart
muscle;
Rat isolated
myocytes;
Calcium
Introduction Results obtained by means of 45Ca2+ (Pytkowski et al., 1983), and by means of extracellular Ca2+ sensitive dyes and extracellular Ca2 + sensitive microelectrodes (Bers, 1983; Bers, 1987; Hilgemann, 1986a, b; Hilgemann and Noble, 1987; Hilgemann et al., 1983; Shattock and Bers, 1989) have established that there is a net Ca2+ influx into the guineapig and rabbit cardiac myocytes during cell activation. Under the steady state conditions the transsarcolemmal Ca2+ influx is compensated by net Ca2+ efflux during later phases of the cell activation (Hilgemann and Noble, 1987; Powell and Noble, 1989) and/or between the beats (Shattock and Bers, 1989). Hence it may be accepted that the activated
influx;
11122 I + 09 $03.00/O
coupling.
sarcolemmal Ca2+ channels provide the main route of Ca2+ influx in these species and that the net Ca2 ’ loss from the cells occurs during diastole. Together with the sodium pump lag promoting Na+ and Ca2+ retention (Langer, 1983; Lewartowski and Pytkowski, 1987) this pattern of Ca 2+ fluxes accounts for positive staircase and rest decay of contractions (Lewartowski et al., 1984; Lewartowski and Pytkowski, 1987; Pytkowski, 1989). Ventricular myocytes of rat differ from those of many other mammals in that they show negative staircase and sustained rest potentiation of contraction. Recently Shattock and Bers (1989) have demonstrated that in the rat ventricular muscle in contrast to that of rabbit, contraction is accompanied by the net cellular Ca2+ efflux,
Please address all correspondence to: B. Lewartowski, Department Postgraduate Education, Marymoncka 99, 0 l-8 13 Warsaw, Poland. This work was supported by the grant No. I 1.6.58.1 of the National 0022-2828/90/
Excitation-contraction
of Clinical Institute
Physiology,
of Cardiology.
Medical
Centrr
Warsaw.
i:, 1990 Acadcmi(
Prrss I.irnited
01
1222
B. Lewartowski
and K. Zdanowski
and that there is a net cellular Ca’+ influx at rest. These authors also found that the intracellular Na+ activity is significantly higher in rat than in the rabbit ventricle. These results led to the hypothesis that Cat+ influx through the Na/Ca exchange occurs in rat ventricular myocytes during diastole and that Ca2+ extrusion is strongly favoured during activation due to the early repolarisation which is complete before the intracellular Ca2+ transient has subsided. Therefore a major influx of Ca2+ into rat myocytes would occur during diastole via Na/Ca exchange. The Ca2+ would be taken up by sarcoplasmic reticulum (SR) and releasedduring the next excitation. This model would explain negative staircase and sustained post-rest potentiation of contractions in this species. In this paper we tested this hypothesis by comparing in single isolated rat and guineapig cardiac myocytes restitution of contraction and repletion of SR Ca2+ after it has been depleted by short application of caffeine in large concentration. The cells respond to caffeine injected into their close vicinity with a strong transient contracture (DuBell, 1988; Stern et al., 1988), apparently due to rapid releaseof Ca2+ from the SR (for review of the mode of action of caffeine seeLewartowski et al., 1990). The Ca2+ released by caffeine is removed from the cell (Callewaert et al., 1989) resulting in strong inhibition of contractile responseto a second injection of caffeine and of the amplitude of post-caffeine contractions (Lewartowski et al., 1990). We found that contractile responsesto the secondinjection of caffeine and post-caffeine contractions recover and SR Ca2+ is promptly repleted in resting rat myocytes but not in thoseofguinea-pig. In rat repletion of SR Ca2+ is completely inhibited by removal of extracellular Ca2 + , by 5.0 mM Ni2+ in superfusate or by hyperpolarising of the cells with low K+ solution. Repletion is enhanced by low Naf solution. The results are consistent with the hypothesis that there is a substantial net Ca2+ influx into the rat ventricular myocytes at rest, but not in the resting guinea-pig cells. Methods
Experiments were performed on isolated, single myocytes of the ventricles of rat and
guinea-pig hearts. The animals were injected i.p. with 1000 U of heparin followed 30 min later by an overdose of nembutal. The hearts were quickly removed and perfused under constant flow conditions using the Langendorffmethod at 37.0%. Isolation of the cells Guinea-pig hearts were perfused for 3 min with the nominally Ca2+ free solution (composition below) followed by ‘20 min of perfusion with the samesolution containing 40 mg of collagenase (Sigma) and 2 mg of protease (Type XIV, No. b-5147, Sigma) per 50 ml. For the last 5 min the hearts were perfused with the Tyrode solution containing 0.2 mM Ca’+. The procedur e for isolation of the rat myocytes was similar, however, protease was not used. After completion of perfusion the ventricles were placed in the beaker containing 50 ml of the Tyrode solution (0.2 mM CaZf), epicardium was disrupted with pincettes and the muscle gently agitated. The dispersed cells were filtered through the nylon mesh and Ca2+ concentration increased to 1.OmM. The final suspensioncontained about 20-50% of the rod-shaped, excitable guinea-pig cells or 50-80% of viable rat cells. Solutions
The modified Tyrode’s solution of the following composition was used (in mM): NaCl 144.0; KC1 5.0; MgCl2 1.0; NaH2P04 0.43; HEPES 10.0; glucose 11.O; CaCla was added as indicated in Methods and Results. The pH of the solution was adjusted with NaOH to 7.3 (for isolation of the cells) or to 7.4 (for experiments), Perfusion of the hearts was performed at 37.O”C, the experiments were performed at the room temperature (about 24°C). Superfusion and stimulation of the cells The cellswere placed in the superfusion chamber and became attached to its glassbottom. The construction of the chamber secured the uni-directional flow of superfusate.The stimulating platinum wire electrodesplaced closeto the bottom of the chamber along its walls enabled the field-stimulation of the myocytes. In other experiments the cells were placed in
Ca In&as
in Rat
Cardiac
the plastic dish (diameter 6.0 cm, Falcon, negatively charged). After selecting the cells the rapid super-fusion system similar to that described by Rich et al., (1988) was placed just above the bottom of the dish. The system was built of an inflow tube (length 5 mm, diameter 1 mm) connected to the outflow of a small electromagnetic valve. A similar suction tube was placed 5 mm apart. Two platinum stimulating electrodes were placed on both sides of the line joining the centres of the tubes. When the system was operating the only solution in the dish was that flowing rapidly between the inflow and outflow tubes. The valve could exchange rapidly the solutions flowing from 2 reservoirs. The valve was activated by the pulse of a stimulator through the adjustable delay circuit.
Caffeine contractures
Myocytes
Results Recovery of post-cafleine contractile responses in the resting rat myocytes
The cells were stimulated at the rate of 20-30/min. Injection of caffeine into the vicinity of the cell instead of an omitted steady state electrical stimulus initiated a transient contracture the amplitude of which attained 175 & 16% (mean & s.D., n = 18) of that of steady state electrically evoked contraction (EEC). [Fig. l(a)]. A second caffeine injection applied 2-5 s after the first one initiated a small contracture, the amplitude of which attained about 30% of that of the first contracture [Fig. 1(a)]. The amplitude of the first post-caffeine EEC was decreasedto 8 f 3:/, of that ofsteady state EEC [Fig. 1(b)]. Recovery of contractile amplitude followed upon steady state stimulation [Fig. 1(b)]. These results show that caffeine releasesmost of SR Ca2+ and that there is little or no immediate re-
In both groups of experiments the tip of a glass micropipette (diameter about 2 pm) was placed about 10 pm from the cell downstream in respect to the flow of superfusing solution. The micropipette was connected to a pneumatic ejecting system. Its valve was activated by the pre-pulse from a stimulator. Hence several pl of the pipette contents could be injected into close vicinity of the cell exactly instead of the omitted stimulating pulse. The pipettes were filled with the Tyrode solution containing 10.0 mM (rats) or 15 mr+4(guineapigs) caffeine. The cells responded to the pulse of caffeine of 1000 ms duration with a transient contracture. Its amplitude was regarded as an indirect index of the total SR Ca2+ content.
Recording of the cell length
The superfusion chamber or plastic dish were placed on the stage of an inverted microscope. The image of the cell was projected from the lens directly onto a TV camera placed below. The camera was connected to the modified video edge tracking system (Rich et al., 1988) enabling the monitoring of cell length. The signalswere recorded by means of the Mark VII chart recorder. For clarity protocols will be described in Results.
1223
(a)
(b)
,,/t”
100
tt
t
Ios
FIGURE 1. Time-dependent recovery of caffeine contracture after the first application ofcaffeine (a) and of the first post-caffeine electrically evoked contraction (b) of the isolated single myocyte of the rat ventricle. The numbers between the panels a and b show delay between the first and the second application of caffeine (a) and between application of caffeine and the first post-caffeine contraction. The arrows point the moment of injection of caffeine into the close vicinity of the cell. The scale to the left-shortening of the cell in percentage of the diastolic cell length. Caffeine contractwe in the bottom panel cxceded the amplifier range.
1224
B. Lewartowski
and K. Zdanowski phenomena and depend either on intracellular redistribution or on influx of Ca2+ from the extracellular space. Therefore we examined these possibilities in the next series of experiments by investigating the effect of Ca2+ free solution and of Ni2+ on post-rest potentiation of EEC and on restitution of caffeine contractures and of post-caffeine EEC.
I
0 f
i IO
0
0 30
60 s
Coffme
FIGURE 2. Contractile response to caffeine (circles, dots) and to the electrical stimulus (squares) applied after the first injection ofcaffeine as a function of time. Caffeine contractures expressed as a percentage of the amplitude of the contractile response to the first application of caffeine. Contractions expressed as a percentage of the amplitude of the pre-caffeine steady state contractions. Filled symbols, broken line-rat myocytes (mean f S.D. n = 9), open symbols, continuous line-guinea-pig myocytes (n = 11).
uptake of the released Ca2+ by SR. When the delay between the first and the second application of caffeine was gradually increased while the cell remained quiescent, the amplitude of the second caffeine contracture increased attaining within 60s almost 100% of amplitude of the first contracture [Fig. 1 (a) and Fig. 21. An increase in delay between the injection of caffeine and the next EEC also resulted in gradual recovery of its amplitude. However, within 60 s it attained only about 40% of the amplitude of pre-caffeine EEC [Fig. 1 (b) and Fig. 21. Comparison of the top and bottom panel of Figure 1 (b) shows that stimulation largely accelerated recovery of post-caffeine contractions. These results show that SR of a rat myocyte depleted of Ca2+ can reaccumulate its Ca2+ store even if the cell is not stimulated. As shown in panel (a) of Figure 3, rest results in the rat myocytes in potentiation of the postrest EEC. Since the amplitude of the post-rest caffeine contracture is also increased (insets in panel (a), Fig. 3), potentiation of EEC apparently results from enhanced accumulation of SR Ca2+ during rest. Repletion of SR Ca2+ depleted by caffeine and post-rest potentiation of EEC apparently are related
The eflect of Ca2+ free solution and of .Ni2+ on post-rest potentiation of EEC and on restitution of caJeine contractures and of post caJeine EEC in the resting rat myocytes. Figure 3(c) and Figure 4(a) illustrate that superfusion of Ca 2+ free solution or Ni2 + in 5.0 mM concentration resulted in immediate inhibition of the contractile response to the next steady state electrical stimulus. The response reappeared immediately upon reperfusion of the normal Tyrode solution. This result conforms well to that of Rich et al. (1988) in that extracellular Ca2+ is necessary to activate contraction and proves that solutions were completely exchanged between the two steady state EECs. Panels (b) and (e) of Figure 3 illustrate that Ca2+ free solution super-fused from the first until the last second of rest resulted in inhibition of post-rest potentiation of EEC. In some cells the amplitude of initial post-rest EEC was strongly reduced. Panel (g) of Figure 3 shows that the Ca2+ free solution superfused throughout the delay between the first and the second application of caffeine also completely inhibited recovery of the second contractile response. The effects of the Ca2+ free solution may depend on prevention of Ca2+ influx from extracellular space, as well as, on enhancing Ca2+ efflux from the cells. Therefore in the next series of experiments we used 5.0 mM Ni2+ instead of the CaZf free solution. Ni2+ has been shown to inhibit the Na/Ca exchange current (Callewaert et al., 1989; Kimura et al., 1987). Hence it may be expected to block Ca2+ flux via Na/Ca exchange in both directions. Panels (c) and (e) ofFigure 4 show that Ni2+ superfused throughout the rest period completely inhibits post-rest potentiation, as well as, restitution of EEC after application of
Ca Idux
in Rat
Cardiac
Myocytes
1LE.5 b
1 dl
e
FIGURE 3. The effect of Ca 2+ free solution (horizontal bars) on post-rest potentiation [(a!, (b), id). IC’] and on recovery of caffeine contractures after the first application of caffeine if, g) in the single isolated myocyte of the rat ventricle. The arrows point the moment of injection ofcaffeine (10 mM) into the close vicinity of the myocyte, Insets in la) show the contractile responses of the myocyte to caffeine applied instead of the last electrical stimulus before rest and the first stimulus after rest. (c) CCa’+ free solution p erfused during steady state stimulation.
caffeine. Panel (g) of Figure 4 shows that Ni2 + superfused throughout the delay between the first and the second application of caffeine completely prevents recovery of the second contractile response. Washout of Ni2+ results in immediate resumption of recovery of contractile response to the next application of caffeine [Fig. 4(g)]. Superfusion of Ni2+ before and during caffeine contracture initiated instead of steady state EEC resulted in slight increase in its amplitude and in delay of relaxation [Fig. 4(h)].
The eflect of low Na’ solution or of low ICC .rolution on restitution of cafleine contractures in resting rat myocytes. The above experiments show that post-rest potentiation and restitution of post-caffeine contractile responses depend on influx of Ca’+ from the extracellular space. The effect of Ni’+ points to Na/Ca exchange as a likely route. In order to test further this possibility we investigated the effect of low Na+ solution with Li+ I or of low K+ (5Oq6, substituted
B. Lewartowski
and K. Zdanowski
(b)
NI 5mM
NI 5mM
id)
lh)
(Q)
20 5
N15rn~
NI
FIGURE 4. The effect of Ni2+ (horizontal bars) on the steady state contractions (a), on post-rest potentiation of contraction (b, c), recovery of post-caffeine electrically evoked contractions (d, e) and on recovery of caffeine contractures after the first application of caffeine (f, g) in the single myocyte of the rat ventricle. Arrows point the moment of injection of caffeine (10 ITIM) into the close vicinity of the cell. (h)-effect of Ni*+ on caffeine contracture evoked instead of steady state contraction.
solution (1.0 mM) on restitution of caffeine contractures. Panel (b) of Figure 5 shows that the low Na+ solution superfiused throughout the delay between the first and the second application of caffeine accelerates restitution of the second contractile response. This intervention is expected to increase the Ca2+ influx via Na/Ca exchange. Lower concentrations of Na+ or longer super-fusion with low Na+ solution resulted in appearance of many spontaneous contractile waves. Low K+ solution super-fused between the two steady state beats resulted in immediate decrease of the amplitude of the next EEC by about 50%. Despite continued superfusion with this solution contractile amplitude slowly recovered reaching the control level within
30-60 s. Superfusion of the normal solution resulted in an immediate increase of the amplitude of EEC by about 50% of control. This potentiation subsided over the next several beats (not shown). These changes apparently resulted from two effects of the low K+ solution: hyperpolarising of the cells which increased the driving force of Na+ and inhibition ofNa+, K+ ATPase enhancing the Na+ accumulation. Therefore in the next experiments we applied only the short (about 10 s) superfiusion with the low K+ solution when the first effect apparently prevailed. Panel (d) of Figure 5 shows that the low K+ solution super-fused between the two applications of caffeine completely inhibits recovery of the second contractile response.
CP Idus
h
i
(a)
n
Cc)
n
in
Rat Cardiac
, 9’) ii j
Myocytes
Ib)
(d)
FIGURE 5. The effect of low Na+ solution (72 m, substituted with Li+) (b) or of low (1 mm) K+ solution (d) on restitution of contractile response to caffeine (arrows) injected into the close vicinity of the single rat cardiomyocyte
Guinea-pig
myoGytes
Injection of caffeine instead of an omitted steady state electrical stimulus initiated a transient contracture, the amplitude of which attained 124 f 12 o/oof that of the steady state EEC. The amplitude of the first post-caffeine EEC decreased to 16 + 6 y. of that of the steady state contractions. Recovery of precaffeine amplitude was completed over several subsequentbeats [Fig. 6(b)]. Increase in delay between the caffeine contracture and the next EEC to 60 s did not result in any recovery of its amplitude [Fig. 6(b)]. The secondinjection of caffeine immediately after the first one did not evoke any contractile response.The response did not recover when the delay between the first and the second caffeine injection was increased upto 60 s [Fig. 6(a)]. Lack of any contractile responseto the second application of caffeine shows that there is no recirculation of Ca’+ under these conditions. Hence the first post-caffeine contraction must have been activated exclusively by extracellular Ca’+ . Short (15-30 s) periods of rest resulted in potentiation of the first post-rest EEC. The second EEC was attenuated in respect to the pre-rest contractions. Pre-rest amplitude of EEC was reached over the few post-rest beats [Fig. 7(b, e)]. Superfusion with Ca’+ free solution or with solution containing 5.0 mM Ni’+ resulted in immediate inhibition of con-
FIGURE 6. Contractions of a single guinea-pig myocyte. Lack of recovery of electrically evoked contractions (b) and of contractile response to caffeine after the first caffeine application (a, arrows). Numbers between the panels a and b show delay before the lint and second application of caffeine (a) or between application of caffeine and the first post-caffeine contraction (b).
tractile responseto the next electrical stimulus. The response reappeared immediately upon reperfusion of the normal Tyrode solution [Fig. 7(a, d)]. Calcium free solution or Ni2+ superfused from the first until the last second of rest only slightly decreased amplitude of the first post-rest EEC asshown by the representative records in Figure 7 (c, f) . Discussion
These results illustrate that the SR in rat myocytes depleted of Ca2+ by means of short exposure to high concentration of caffeine, in contrast to SR in guinea-pig myocytes, is able to reaccumulate Ca2+ even if the cell is not stimulated. This was shown by the resting restitution of post-caffeine EEC and by restitution of contractile response to the second application of caffeine (Fig. 1). These properties of the rat myocytes could be explained either by the rest-dependent shifts of Ca’ + between the intracellular compartments or by a resting Ca2+ influx asproposed by Shattock and Bers (1989). Post-rest potentiation and restitution of post-caffeine contractile responseswere completely inhibited by removal
ca Influx iu Rat cardiac
1228
Myocytes
(b)
Cd)
NI 5mM
(e)
IO 5
NiSmM
FIGURE 7. Contractions of a single guinea-pig myocyte. The effect of no Ca’+ solution (b, c) and Ni2+ (e, f) on post-rest potentiation of contractions. (a). cd)-immediate inhibition of steady state contraction by no Ca*+ solution and Ni* + , respectively.
of extracellular Ca2+ during the rest period (Fig. 3). This intervention prevents Ca2+ influx from the extracellular space but it may also be expected to enhance Ca2 + efflux. Strong inhibition of the first post-rest contraction by superfusion with Ca2+ free solution observed in somecells suggeststhat this might be the case. However, post-rest potentiation and restitution of post-caffeine contractile responseswere also completely inhibited by 5.0 mM Ni2+ (Fig. 4). This concentration of Ni2+ was shown to inhibit the Na/Ca exchange current (Kimura et al., 1987; Callewaert et al., 1989) and to block both T and L calcium channels (Mitra and Morad, 1986). Hence Ni2+ may block Ca2+ influx into the resting rat myocytes by means of Na/Ca exchange as proposed by Shattock and Bers (1989) and by meansof T Ca2+ channels if they are partially activated at rest. Ni2+ did not affect the amplitude of the caffeine contracture initiated instead of the last steady state electrical stimulus i.e. when SR was loaded with Ca’+. Relaxation was slowed down. These results show that post-rest potentiation and restitution of post-caffeine contractile responsesdepend on influx of extracellular Ca2+ into the resting
Na/Ca exchange, Panel (b) of Figure 5 illustrates that superfusion with the low Na+ solution enhances restitution of contractile responseto the second application of caffeine. Reduction of K+ concentration from 5.0 mM to 1.OmM is expected to hyperpolarise the cells thus increasing the driving force of Na+ and shifting the resting membrane potential towards or below (more negative) the Na/Ca reversal potential (Mullins, 1979). Thus hyperpolarisation should decrease the Ca2+ influx or even promote the Ca2+ e&x via the Na/Ca exchange. Panel (d) of Figure 5 shows that superfusion with the low K+ solution strongly inhibits the restitution of contractile responseto the second application of caffeine. We feel that experiments with Ni*+, low Na+ solution or low K+ solution have proved a strong link of the Ca2+ influx into the resting rat myocytes to Na/Ca exchange. In guinea-pig potentiation of the first contraction after short rest was only slightly diminished by Ca2+ free solution or by Ni2 + superfusedduring break in stimulation. These results suggestthat in guinea-pig potentiation results exclusively from Ca2+ shifts between the cellular compartments at rest. No restirat myocyte. tution of the post-caffeine EEC or of contracThe dependence of the Ca2+ influx on tile responseto the secondcaffeine application Na/Ca exchange was further tested by means was observed unless the cell was stimulated. of superfusionof the cells throughout the delay These results show that the only route of between the first and the secondapplication of significant net Ca2+ influx into the guinea-pig caffeine with solution containing 50% of myocytes are the Ca 2+ channels activated at normal Na+ or 1.OmM K+. The first interven- excitation and possibly the Na/Ca exchange tion is expected to decrease the Na’ driving working transiently in the Ca2+ entry mode in force thus enhancing the Ca2+ influx via the the depolarised cell (Lederer et al., 1989; Wier
B. Lewartowski
and K. Zdanowski
and Beuckelmann, 1989; Powell and Noble, 1989). The results of this work are generally consistent with Shattock and Bers’ (1989) hypothesisaccording to which resting Ca*+ influx by meansof Na/Ca exchange is the important means by which the amount of SR Ca*+ is increased during rest in the rat cardiac myocytes. However, asillustrated in Figure 1 (b) the stimulated cells recovered their precaffeine contractile amplitude much quicker than the rested cells. Acceleration of recovery by stimulation could mean that either Ca*+ influx through the activated calcium channels alsoprovides an important source to replenish the SR Ca*+ storesor that Na+ entry through Na+ channels and Na/Ca exchanger operating in the Ca* + extrusion mode during excitation of the cell enhances Na/Ca exchange
I229
operating in the Ca2+ entry mode between the contractions. Figures 1 and 2 show that recovery of amplitude of the post-caffeine EEC lags behind the repletion of the SR Ca*+ indicated by the amplitude of the caffeine contractures. This discrepancy was not observed after rest of respective duration as shown by the insets in Figure 3 (a) : post-rest potentiation of EEC was accompanied by potentiation of caffeine contractures. The reason for the discrepancy is not clear and requires further investigation. Acknowledgement>
The authors are greatly indebted to professor Glenn A. Langer, M.D. for the generousgift of the video edge tracking systemand for sharing with us his method of rapid cell superfusion.
References BERS DM
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(1983) Early H366-H381.
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of extracellular
[Cal
during
individual
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contractions.
Am J
CALLEWAERT G, CLEEMAN L, MORAD M (1989) Caffeine-induced CazC release activates Ca*+ extrusion via Na+ -Ca*+ exchanger in cardiac myocytes. Am J Physiol, 257, C147-C152. DUBELL WH (1988) The characterisation of the calcium transient in cardiac feline ventricular myocytes using the calcium sensitive fluorescent dye Indo-1. Dissertation, Temple University, Philadelphia, PA. HILGEMANN DW (1986a) Extracellular calcium transients and action potential configuration changes related to poststimulatory potentiation in rabbit atrium. J Gen Physiol87, 675-706. HILGEMANN DW (1986b) Extracellular calcium transients at single contractions in rabbit atrium measured with tetramethylmurexide. J Gen Physio187, 707-735. HILGEMANN DW, DELAY MJ, LANCER GA (1983) Activation-dependent cumulative depletions of extracellular free calcium in guinea-pig atrium measured with antipyrylazo III and tetramethylmurexide. Circ Res 53, 779-793. HILGEMANN DW, NOBLE D (1987) Excitation-contraction coupling and extracellular calcium transients in rahbit atrium: reconstruction of basic cellular mechanism. Proc Roy Sot B, 230, 163-205. KIMURA J, MIYAMA S, NOMA A (1987) Identification of sodium-calcium exchange current in single ventricular cells of guinea-pig. J Physiol [Lond] 384, 199-222. LANCER GA (1983) The “sodium pump lag” revisited. J Mol Cell Cardiol 15, 64745 1. LEDERER WJ, CANNELL MB, COHEN NM, BERLIN JR (1989) Excitation-contraction coupling in heart muscle. Mol Cell Biochem 89, 115-l 19. LEWARTOWSKI B, PYTKOWSKI B., JANCZEWSKI A (1984) Calcium fraction correlating with contractile force ofventricular muscle of guinea-pig heart. Pfliigers Arch 44)1, 198-203. LEWARTOWSKI B, HANSFORD RG, LANCER GA, LAKATTA EG [in press) Excitation-contraction coupling and sarcoplasmic reticulum Ca*+ content in single myocytes of guinea-pig heart: the effect of ryanodine. Am J Physiol. MITRA R, MORAD M (1986) Two types of calcium channels in guinea-pig ventricular myocytes. Proc Nat1 Acad Sci USA 83, 5340-5344. MULLINS LI (1979) The generation of electric currents in cardiac fihres by Na/Ca exchange. Am J Physiol 236, C103-c110. POWELL T, NOBLE D (1989) Calcium movements during each heart beat. Mol Cell Biochem 89, 103-108. PYTKOWSKI B, LEWARTOWSKI B, PROKOPCZUK A, ZDANOWSKI K, LEWANDOWSKA K 11983) Excitationand rcstdependent shifts of Ca in guinea-pig ventricular myocardium. Pfltigers Arch, 398, 103-l 13. RICH TI, LANCER GA, KLASSEN MG (1989). Two components of coupling calcium in single ventricular cells of rabbits and rats. Am J Physiol, 254, H937-H946. SHATTOCK MJ, BERS DM (1989) Rat vs rabbit ventricle: Ca flux and intracellular Na assessed by ion-sclrrtivr microelectrodes. Am J Physiol, 256, C813-C822. STERN MD, SILVERMAN HS, HOUSER SR, JOSEPHSON MC, CAPOGROSSI CG, NICHOLS WJ, LEDERER WJ. LAKATTA EC 1988) Anoxic contractile failure in rat heart myocytes is caused by failure of intracellular calcium release dur trl alteration of action potential. Proc Nat1 Acad Sci USA, 85, 695&6958. WIER WG, BEUCKELMANN DJ (1989) Sodium-calcium exchange in mammalian heart: current-voltage rcalation and intracellular calcium concentration. Mol Cell Biochem, 89, 97-102.
Cell
Cardiol
22,
1221-1229
(1990)
Net Ca2 + Infhx and Sarcoplasmic Reticulum Ca2 + Uptake in Resting Single Myocytes of the Rat Heart: Comparison with Guinea-pig Bohdan
Lewartowski
and Krzysstof
Zdanowski
Department of Clinical Physiology, Medical Center of Postgraduate Education, Warsaw, Poland (Received 22 January 1990, accepted in revisedform
8 May 1990)
B. LEWARTOWSKI AND K. ZDANOWSKI. Net Ca2+ Influx and Sarcoplasmic Reticulum Ca2+ Uptake in Resting Single Myocytes of the Rat Heart: Comparison with Guinea-pig. Journal of Molecular and Cellular Cardiology (1990) 22, 1221-1229. We tested Shattock and Bers’ (1989) hypothesis according to which in rat cardiac myocytes net Ca2’ influx during diastole via Na/Ca exchange provides the main route of entry of Ca’+ available for activation ofcontractions. We used injections ofcaffeine into the close vicinity of the single, isolated rat or guinea(SR). The cells responded to pig ventricular myocytes in order to release Ca’+ from sarcop lasmic reticulum caffeine with a transient contracture, the amplitude of which was regarded as a relative index of SR Ca2+ content. Application of caffeine deprived the SR of Ca 2+ This was manifested by a very small (rat) or absent (guinea-pig) contractile response to the second application of caffeine and by a decrease of the amplitude of the first post caffeine contraction to 8 + 3% (rat) or to 16 f Sy/, (guinea-pig) of control. In the rat myocytes SR deprived of Ca2+ was able to recover its Ca2+ store even in the resting cell. This was indicated by the time dependent recovery of contractile response to the second application of caffeine and of the amplitude of the postcaffeine electrically evoked contractions. The recovery of post-caffeine contractile responses was completely inhibited by Ca2+ free solution, by 5.0 mM NiC and by low K+ (1 .O mM) hyperpolarising solution superfused from the first application of caffeine or during rest. The recovery was enhanced by superfusion of the cells with low Na+ (50%) solution. These results show that there is a considerable net Ca*+ influx by means of Na/Ca exchange and then the SR Ca*+ uptake in the resting rat myocytes. No recovery of post caffeine contractile responses could be observed in guinea-pig myocytes. KEY
WORDS:
Heart
muscle;
Rat isolated
myocytes;
Calcium
Introduction Results obtained by means of 45Ca2+ (Pytkowski et al., 1983), and by means of extracellular Ca2+ sensitive dyes and extracellular Ca2 + sensitive microelectrodes (Bers, 1983; Bers, 1987; Hilgemann, 1986a, b; Hilgemann and Noble, 1987; Hilgemann et al., 1983; Shattock and Bers, 1989) have established that there is a net Ca2+ influx into the guineapig and rabbit cardiac myocytes during cell activation. Under the steady state conditions the transsarcolemmal Ca2+ influx is compensated by net Ca2+ efflux during later phases of the cell activation (Hilgemann and Noble, 1987; Powell and Noble, 1989) and/or between the beats (Shattock and Bers, 1989). Hence it may be accepted that the activated
influx;
11122 I + 09 $03.00/O
coupling.
sarcolemmal Ca2+ channels provide the main route of Ca2+ influx in these species and that the net Ca2 ’ loss from the cells occurs during diastole. Together with the sodium pump lag promoting Na+ and Ca2+ retention (Langer, 1983; Lewartowski and Pytkowski, 1987) this pattern of Ca 2+ fluxes accounts for positive staircase and rest decay of contractions (Lewartowski et al., 1984; Lewartowski and Pytkowski, 1987; Pytkowski, 1989). Ventricular myocytes of rat differ from those of many other mammals in that they show negative staircase and sustained rest potentiation of contraction. Recently Shattock and Bers (1989) have demonstrated that in the rat ventricular muscle in contrast to that of rabbit, contraction is accompanied by the net cellular Ca2+ efflux,
Please address all correspondence to: B. Lewartowski, Department Postgraduate Education, Marymoncka 99, 0 l-8 13 Warsaw, Poland. This work was supported by the grant No. I 1.6.58.1 of the National 0022-2828/90/
Excitation-contraction
of Clinical Institute
Physiology,
of Cardiology.
Medical
Centrr
Warsaw.
i:, 1990 Acadcmi(
Prrss I.irnited
01
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B. Lewartowski
and K. Zdanowski
and that there is a net cellular Ca’+ influx at rest. These authors also found that the intracellular Na+ activity is significantly higher in rat than in the rabbit ventricle. These results led to the hypothesis that Cat+ influx through the Na/Ca exchange occurs in rat ventricular myocytes during diastole and that Ca2+ extrusion is strongly favoured during activation due to the early repolarisation which is complete before the intracellular Ca2+ transient has subsided. Therefore a major influx of Ca2+ into rat myocytes would occur during diastole via Na/Ca exchange. The Ca2+ would be taken up by sarcoplasmic reticulum (SR) and releasedduring the next excitation. This model would explain negative staircase and sustained post-rest potentiation of contractions in this species. In this paper we tested this hypothesis by comparing in single isolated rat and guineapig cardiac myocytes restitution of contraction and repletion of SR Ca2+ after it has been depleted by short application of caffeine in large concentration. The cells respond to caffeine injected into their close vicinity with a strong transient contracture (DuBell, 1988; Stern et al., 1988), apparently due to rapid releaseof Ca2+ from the SR (for review of the mode of action of caffeine seeLewartowski et al., 1990). The Ca2+ released by caffeine is removed from the cell (Callewaert et al., 1989) resulting in strong inhibition of contractile responseto a second injection of caffeine and of the amplitude of post-caffeine contractions (Lewartowski et al., 1990). We found that contractile responsesto the secondinjection of caffeine and post-caffeine contractions recover and SR Ca2+ is promptly repleted in resting rat myocytes but not in thoseofguinea-pig. In rat repletion of SR Ca2+ is completely inhibited by removal of extracellular Ca2 + , by 5.0 mM Ni2+ in superfusate or by hyperpolarising of the cells with low K+ solution. Repletion is enhanced by low Naf solution. The results are consistent with the hypothesis that there is a substantial net Ca2+ influx into the rat ventricular myocytes at rest, but not in the resting guinea-pig cells. Methods
Experiments were performed on isolated, single myocytes of the ventricles of rat and
guinea-pig hearts. The animals were injected i.p. with 1000 U of heparin followed 30 min later by an overdose of nembutal. The hearts were quickly removed and perfused under constant flow conditions using the Langendorffmethod at 37.0%. Isolation of the cells Guinea-pig hearts were perfused for 3 min with the nominally Ca2+ free solution (composition below) followed by ‘20 min of perfusion with the samesolution containing 40 mg of collagenase (Sigma) and 2 mg of protease (Type XIV, No. b-5147, Sigma) per 50 ml. For the last 5 min the hearts were perfused with the Tyrode solution containing 0.2 mM Ca’+. The procedur e for isolation of the rat myocytes was similar, however, protease was not used. After completion of perfusion the ventricles were placed in the beaker containing 50 ml of the Tyrode solution (0.2 mM CaZf), epicardium was disrupted with pincettes and the muscle gently agitated. The dispersed cells were filtered through the nylon mesh and Ca2+ concentration increased to 1.OmM. The final suspensioncontained about 20-50% of the rod-shaped, excitable guinea-pig cells or 50-80% of viable rat cells. Solutions
The modified Tyrode’s solution of the following composition was used (in mM): NaCl 144.0; KC1 5.0; MgCl2 1.0; NaH2P04 0.43; HEPES 10.0; glucose 11.O; CaCla was added as indicated in Methods and Results. The pH of the solution was adjusted with NaOH to 7.3 (for isolation of the cells) or to 7.4 (for experiments), Perfusion of the hearts was performed at 37.O”C, the experiments were performed at the room temperature (about 24°C). Superfusion and stimulation of the cells The cellswere placed in the superfusion chamber and became attached to its glassbottom. The construction of the chamber secured the uni-directional flow of superfusate.The stimulating platinum wire electrodesplaced closeto the bottom of the chamber along its walls enabled the field-stimulation of the myocytes. In other experiments the cells were placed in
Ca In&as
in Rat
Cardiac
the plastic dish (diameter 6.0 cm, Falcon, negatively charged). After selecting the cells the rapid super-fusion system similar to that described by Rich et al., (1988) was placed just above the bottom of the dish. The system was built of an inflow tube (length 5 mm, diameter 1 mm) connected to the outflow of a small electromagnetic valve. A similar suction tube was placed 5 mm apart. Two platinum stimulating electrodes were placed on both sides of the line joining the centres of the tubes. When the system was operating the only solution in the dish was that flowing rapidly between the inflow and outflow tubes. The valve could exchange rapidly the solutions flowing from 2 reservoirs. The valve was activated by the pulse of a stimulator through the adjustable delay circuit.
Caffeine contractures
Myocytes
Results Recovery of post-cafleine contractile responses in the resting rat myocytes
The cells were stimulated at the rate of 20-30/min. Injection of caffeine into the vicinity of the cell instead of an omitted steady state electrical stimulus initiated a transient contracture the amplitude of which attained 175 & 16% (mean & s.D., n = 18) of that of steady state electrically evoked contraction (EEC). [Fig. l(a)]. A second caffeine injection applied 2-5 s after the first one initiated a small contracture, the amplitude of which attained about 30% of that of the first contracture [Fig. 1(a)]. The amplitude of the first post-caffeine EEC was decreasedto 8 f 3:/, of that ofsteady state EEC [Fig. 1(b)]. Recovery of contractile amplitude followed upon steady state stimulation [Fig. 1(b)]. These results show that caffeine releasesmost of SR Ca2+ and that there is little or no immediate re-
In both groups of experiments the tip of a glass micropipette (diameter about 2 pm) was placed about 10 pm from the cell downstream in respect to the flow of superfusing solution. The micropipette was connected to a pneumatic ejecting system. Its valve was activated by the pre-pulse from a stimulator. Hence several pl of the pipette contents could be injected into close vicinity of the cell exactly instead of the omitted stimulating pulse. The pipettes were filled with the Tyrode solution containing 10.0 mM (rats) or 15 mr+4(guineapigs) caffeine. The cells responded to the pulse of caffeine of 1000 ms duration with a transient contracture. Its amplitude was regarded as an indirect index of the total SR Ca2+ content.
Recording of the cell length
The superfusion chamber or plastic dish were placed on the stage of an inverted microscope. The image of the cell was projected from the lens directly onto a TV camera placed below. The camera was connected to the modified video edge tracking system (Rich et al., 1988) enabling the monitoring of cell length. The signalswere recorded by means of the Mark VII chart recorder. For clarity protocols will be described in Results.
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(a)
(b)
,,/t”
100
tt
t
Ios
FIGURE 1. Time-dependent recovery of caffeine contracture after the first application ofcaffeine (a) and of the first post-caffeine electrically evoked contraction (b) of the isolated single myocyte of the rat ventricle. The numbers between the panels a and b show delay between the first and the second application of caffeine (a) and between application of caffeine and the first post-caffeine contraction. The arrows point the moment of injection of caffeine into the close vicinity of the cell. The scale to the left-shortening of the cell in percentage of the diastolic cell length. Caffeine contractwe in the bottom panel cxceded the amplifier range.
1224
B. Lewartowski
and K. Zdanowski phenomena and depend either on intracellular redistribution or on influx of Ca2+ from the extracellular space. Therefore we examined these possibilities in the next series of experiments by investigating the effect of Ca2+ free solution and of Ni2+ on post-rest potentiation of EEC and on restitution of caffeine contractures and of post-caffeine EEC.
I
0 f
i IO
0
0 30
60 s
Coffme
FIGURE 2. Contractile response to caffeine (circles, dots) and to the electrical stimulus (squares) applied after the first injection ofcaffeine as a function of time. Caffeine contractures expressed as a percentage of the amplitude of the contractile response to the first application of caffeine. Contractions expressed as a percentage of the amplitude of the pre-caffeine steady state contractions. Filled symbols, broken line-rat myocytes (mean f S.D. n = 9), open symbols, continuous line-guinea-pig myocytes (n = 11).
uptake of the released Ca2+ by SR. When the delay between the first and the second application of caffeine was gradually increased while the cell remained quiescent, the amplitude of the second caffeine contracture increased attaining within 60s almost 100% of amplitude of the first contracture [Fig. 1 (a) and Fig. 21. An increase in delay between the injection of caffeine and the next EEC also resulted in gradual recovery of its amplitude. However, within 60 s it attained only about 40% of the amplitude of pre-caffeine EEC [Fig. 1 (b) and Fig. 21. Comparison of the top and bottom panel of Figure 1 (b) shows that stimulation largely accelerated recovery of post-caffeine contractions. These results show that SR of a rat myocyte depleted of Ca2+ can reaccumulate its Ca2+ store even if the cell is not stimulated. As shown in panel (a) of Figure 3, rest results in the rat myocytes in potentiation of the postrest EEC. Since the amplitude of the post-rest caffeine contracture is also increased (insets in panel (a), Fig. 3), potentiation of EEC apparently results from enhanced accumulation of SR Ca2+ during rest. Repletion of SR Ca2+ depleted by caffeine and post-rest potentiation of EEC apparently are related
The eflect of Ca2+ free solution and of .Ni2+ on post-rest potentiation of EEC and on restitution of caJeine contractures and of post caJeine EEC in the resting rat myocytes. Figure 3(c) and Figure 4(a) illustrate that superfusion of Ca 2+ free solution or Ni2 + in 5.0 mM concentration resulted in immediate inhibition of the contractile response to the next steady state electrical stimulus. The response reappeared immediately upon reperfusion of the normal Tyrode solution. This result conforms well to that of Rich et al. (1988) in that extracellular Ca2+ is necessary to activate contraction and proves that solutions were completely exchanged between the two steady state EECs. Panels (b) and (e) of Figure 3 illustrate that Ca2+ free solution super-fused from the first until the last second of rest resulted in inhibition of post-rest potentiation of EEC. In some cells the amplitude of initial post-rest EEC was strongly reduced. Panel (g) of Figure 3 shows that the Ca2+ free solution superfused throughout the delay between the first and the second application of caffeine also completely inhibited recovery of the second contractile response. The effects of the Ca2+ free solution may depend on prevention of Ca2+ influx from extracellular space, as well as, on enhancing Ca2+ efflux from the cells. Therefore in the next series of experiments we used 5.0 mM Ni2+ instead of the CaZf free solution. Ni2+ has been shown to inhibit the Na/Ca exchange current (Callewaert et al., 1989; Kimura et al., 1987). Hence it may be expected to block Ca2+ flux via Na/Ca exchange in both directions. Panels (c) and (e) ofFigure 4 show that Ni2+ superfused throughout the rest period completely inhibits post-rest potentiation, as well as, restitution of EEC after application of
Ca Idux
in Rat
Cardiac
Myocytes
1LE.5 b
1 dl
e
FIGURE 3. The effect of Ca 2+ free solution (horizontal bars) on post-rest potentiation [(a!, (b), id). IC’] and on recovery of caffeine contractures after the first application of caffeine if, g) in the single isolated myocyte of the rat ventricle. The arrows point the moment of injection ofcaffeine (10 mM) into the close vicinity of the myocyte, Insets in la) show the contractile responses of the myocyte to caffeine applied instead of the last electrical stimulus before rest and the first stimulus after rest. (c) CCa’+ free solution p erfused during steady state stimulation.
caffeine. Panel (g) of Figure 4 shows that Ni2 + superfused throughout the delay between the first and the second application of caffeine completely prevents recovery of the second contractile response. Washout of Ni2+ results in immediate resumption of recovery of contractile response to the next application of caffeine [Fig. 4(g)]. Superfusion of Ni2+ before and during caffeine contracture initiated instead of steady state EEC resulted in slight increase in its amplitude and in delay of relaxation [Fig. 4(h)].
The eflect of low Na’ solution or of low ICC .rolution on restitution of cafleine contractures in resting rat myocytes. The above experiments show that post-rest potentiation and restitution of post-caffeine contractile responses depend on influx of Ca’+ from the extracellular space. The effect of Ni’+ points to Na/Ca exchange as a likely route. In order to test further this possibility we investigated the effect of low Na+ solution with Li+ I or of low K+ (5Oq6, substituted
B. Lewartowski
and K. Zdanowski
(b)
NI 5mM
NI 5mM
id)
lh)
(Q)
20 5
N15rn~
NI
FIGURE 4. The effect of Ni2+ (horizontal bars) on the steady state contractions (a), on post-rest potentiation of contraction (b, c), recovery of post-caffeine electrically evoked contractions (d, e) and on recovery of caffeine contractures after the first application of caffeine (f, g) in the single myocyte of the rat ventricle. Arrows point the moment of injection of caffeine (10 ITIM) into the close vicinity of the cell. (h)-effect of Ni*+ on caffeine contracture evoked instead of steady state contraction.
solution (1.0 mM) on restitution of caffeine contractures. Panel (b) of Figure 5 shows that the low Na+ solution superfiused throughout the delay between the first and the second application of caffeine accelerates restitution of the second contractile response. This intervention is expected to increase the Ca2+ influx via Na/Ca exchange. Lower concentrations of Na+ or longer super-fusion with low Na+ solution resulted in appearance of many spontaneous contractile waves. Low K+ solution super-fused between the two steady state beats resulted in immediate decrease of the amplitude of the next EEC by about 50%. Despite continued superfusion with this solution contractile amplitude slowly recovered reaching the control level within
30-60 s. Superfusion of the normal solution resulted in an immediate increase of the amplitude of EEC by about 50% of control. This potentiation subsided over the next several beats (not shown). These changes apparently resulted from two effects of the low K+ solution: hyperpolarising of the cells which increased the driving force of Na+ and inhibition ofNa+, K+ ATPase enhancing the Na+ accumulation. Therefore in the next experiments we applied only the short (about 10 s) superfiusion with the low K+ solution when the first effect apparently prevailed. Panel (d) of Figure 5 shows that the low K+ solution super-fused between the two applications of caffeine completely inhibits recovery of the second contractile response.
CP Idus
h
i
(a)
n
Cc)
n
in
Rat Cardiac
, 9’) ii j
Myocytes
Ib)
(d)
FIGURE 5. The effect of low Na+ solution (72 m, substituted with Li+) (b) or of low (1 mm) K+ solution (d) on restitution of contractile response to caffeine (arrows) injected into the close vicinity of the single rat cardiomyocyte
Guinea-pig
myoGytes
Injection of caffeine instead of an omitted steady state electrical stimulus initiated a transient contracture, the amplitude of which attained 124 f 12 o/oof that of the steady state EEC. The amplitude of the first post-caffeine EEC decreased to 16 + 6 y. of that of the steady state contractions. Recovery of precaffeine amplitude was completed over several subsequentbeats [Fig. 6(b)]. Increase in delay between the caffeine contracture and the next EEC to 60 s did not result in any recovery of its amplitude [Fig. 6(b)]. The secondinjection of caffeine immediately after the first one did not evoke any contractile response.The response did not recover when the delay between the first and the second caffeine injection was increased upto 60 s [Fig. 6(a)]. Lack of any contractile responseto the second application of caffeine shows that there is no recirculation of Ca’+ under these conditions. Hence the first post-caffeine contraction must have been activated exclusively by extracellular Ca’+ . Short (15-30 s) periods of rest resulted in potentiation of the first post-rest EEC. The second EEC was attenuated in respect to the pre-rest contractions. Pre-rest amplitude of EEC was reached over the few post-rest beats [Fig. 7(b, e)]. Superfusion with Ca’+ free solution or with solution containing 5.0 mM Ni’+ resulted in immediate inhibition of con-
FIGURE 6. Contractions of a single guinea-pig myocyte. Lack of recovery of electrically evoked contractions (b) and of contractile response to caffeine after the first caffeine application (a, arrows). Numbers between the panels a and b show delay before the lint and second application of caffeine (a) or between application of caffeine and the first post-caffeine contraction (b).
tractile responseto the next electrical stimulus. The response reappeared immediately upon reperfusion of the normal Tyrode solution [Fig. 7(a, d)]. Calcium free solution or Ni2+ superfused from the first until the last second of rest only slightly decreased amplitude of the first post-rest EEC asshown by the representative records in Figure 7 (c, f) . Discussion
These results illustrate that the SR in rat myocytes depleted of Ca2+ by means of short exposure to high concentration of caffeine, in contrast to SR in guinea-pig myocytes, is able to reaccumulate Ca2+ even if the cell is not stimulated. This was shown by the resting restitution of post-caffeine EEC and by restitution of contractile response to the second application of caffeine (Fig. 1). These properties of the rat myocytes could be explained either by the rest-dependent shifts of Ca’ + between the intracellular compartments or by a resting Ca2+ influx asproposed by Shattock and Bers (1989). Post-rest potentiation and restitution of post-caffeine contractile responseswere completely inhibited by removal
ca Influx iu Rat cardiac
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Myocytes
(b)
Cd)
NI 5mM
(e)
IO 5
NiSmM
FIGURE 7. Contractions of a single guinea-pig myocyte. The effect of no Ca’+ solution (b, c) and Ni2+ (e, f) on post-rest potentiation of contractions. (a). cd)-immediate inhibition of steady state contraction by no Ca*+ solution and Ni* + , respectively.
of extracellular Ca2+ during the rest period (Fig. 3). This intervention prevents Ca2+ influx from the extracellular space but it may also be expected to enhance Ca2 + efflux. Strong inhibition of the first post-rest contraction by superfusion with Ca2+ free solution observed in somecells suggeststhat this might be the case. However, post-rest potentiation and restitution of post-caffeine contractile responseswere also completely inhibited by 5.0 mM Ni2+ (Fig. 4). This concentration of Ni2+ was shown to inhibit the Na/Ca exchange current (Kimura et al., 1987; Callewaert et al., 1989) and to block both T and L calcium channels (Mitra and Morad, 1986). Hence Ni2+ may block Ca2+ influx into the resting rat myocytes by means of Na/Ca exchange as proposed by Shattock and Bers (1989) and by meansof T Ca2+ channels if they are partially activated at rest. Ni2+ did not affect the amplitude of the caffeine contracture initiated instead of the last steady state electrical stimulus i.e. when SR was loaded with Ca’+. Relaxation was slowed down. These results show that post-rest potentiation and restitution of post-caffeine contractile responsesdepend on influx of extracellular Ca2+ into the resting
Na/Ca exchange, Panel (b) of Figure 5 illustrates that superfusion with the low Na+ solution enhances restitution of contractile responseto the second application of caffeine. Reduction of K+ concentration from 5.0 mM to 1.OmM is expected to hyperpolarise the cells thus increasing the driving force of Na+ and shifting the resting membrane potential towards or below (more negative) the Na/Ca reversal potential (Mullins, 1979). Thus hyperpolarisation should decrease the Ca2+ influx or even promote the Ca2+ e&x via the Na/Ca exchange. Panel (d) of Figure 5 shows that superfusion with the low K+ solution strongly inhibits the restitution of contractile responseto the second application of caffeine. We feel that experiments with Ni*+, low Na+ solution or low K+ solution have proved a strong link of the Ca2+ influx into the resting rat myocytes to Na/Ca exchange. In guinea-pig potentiation of the first contraction after short rest was only slightly diminished by Ca2+ free solution or by Ni2 + superfusedduring break in stimulation. These results suggestthat in guinea-pig potentiation results exclusively from Ca2+ shifts between the cellular compartments at rest. No restirat myocyte. tution of the post-caffeine EEC or of contracThe dependence of the Ca2+ influx on tile responseto the secondcaffeine application Na/Ca exchange was further tested by means was observed unless the cell was stimulated. of superfusionof the cells throughout the delay These results show that the only route of between the first and the secondapplication of significant net Ca2+ influx into the guinea-pig caffeine with solution containing 50% of myocytes are the Ca 2+ channels activated at normal Na+ or 1.OmM K+. The first interven- excitation and possibly the Na/Ca exchange tion is expected to decrease the Na’ driving working transiently in the Ca2+ entry mode in force thus enhancing the Ca2+ influx via the the depolarised cell (Lederer et al., 1989; Wier
B. Lewartowski
and K. Zdanowski
and Beuckelmann, 1989; Powell and Noble, 1989). The results of this work are generally consistent with Shattock and Bers’ (1989) hypothesisaccording to which resting Ca*+ influx by meansof Na/Ca exchange is the important means by which the amount of SR Ca*+ is increased during rest in the rat cardiac myocytes. However, asillustrated in Figure 1 (b) the stimulated cells recovered their precaffeine contractile amplitude much quicker than the rested cells. Acceleration of recovery by stimulation could mean that either Ca*+ influx through the activated calcium channels alsoprovides an important source to replenish the SR Ca*+ storesor that Na+ entry through Na+ channels and Na/Ca exchanger operating in the Ca* + extrusion mode during excitation of the cell enhances Na/Ca exchange
I229
operating in the Ca2+ entry mode between the contractions. Figures 1 and 2 show that recovery of amplitude of the post-caffeine EEC lags behind the repletion of the SR Ca*+ indicated by the amplitude of the caffeine contractures. This discrepancy was not observed after rest of respective duration as shown by the insets in Figure 3 (a) : post-rest potentiation of EEC was accompanied by potentiation of caffeine contractures. The reason for the discrepancy is not clear and requires further investigation. Acknowledgement>
The authors are greatly indebted to professor Glenn A. Langer, M.D. for the generousgift of the video edge tracking systemand for sharing with us his method of rapid cell superfusion.
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Physiol244,
(1983) Early H366-H381.
transient
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of extracellular
[Cal
during
individual
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contractions.
Am J
CALLEWAERT G, CLEEMAN L, MORAD M (1989) Caffeine-induced CazC release activates Ca*+ extrusion via Na+ -Ca*+ exchanger in cardiac myocytes. Am J Physiol, 257, C147-C152. DUBELL WH (1988) The characterisation of the calcium transient in cardiac feline ventricular myocytes using the calcium sensitive fluorescent dye Indo-1. Dissertation, Temple University, Philadelphia, PA. HILGEMANN DW (1986a) Extracellular calcium transients and action potential configuration changes related to poststimulatory potentiation in rabbit atrium. J Gen Physiol87, 675-706. HILGEMANN DW (1986b) Extracellular calcium transients at single contractions in rabbit atrium measured with tetramethylmurexide. J Gen Physio187, 707-735. HILGEMANN DW, DELAY MJ, LANCER GA (1983) Activation-dependent cumulative depletions of extracellular free calcium in guinea-pig atrium measured with antipyrylazo III and tetramethylmurexide. Circ Res 53, 779-793. HILGEMANN DW, NOBLE D (1987) Excitation-contraction coupling and extracellular calcium transients in rahbit atrium: reconstruction of basic cellular mechanism. Proc Roy Sot B, 230, 163-205. KIMURA J, MIYAMA S, NOMA A (1987) Identification of sodium-calcium exchange current in single ventricular cells of guinea-pig. J Physiol [Lond] 384, 199-222. LANCER GA (1983) The “sodium pump lag” revisited. J Mol Cell Cardiol 15, 64745 1. LEDERER WJ, CANNELL MB, COHEN NM, BERLIN JR (1989) Excitation-contraction coupling in heart muscle. Mol Cell Biochem 89, 115-l 19. LEWARTOWSKI B, PYTKOWSKI B., JANCZEWSKI A (1984) Calcium fraction correlating with contractile force ofventricular muscle of guinea-pig heart. Pfliigers Arch 44)1, 198-203. LEWARTOWSKI B, HANSFORD RG, LANCER GA, LAKATTA EG [in press) Excitation-contraction coupling and sarcoplasmic reticulum Ca*+ content in single myocytes of guinea-pig heart: the effect of ryanodine. Am J Physiol. MITRA R, MORAD M (1986) Two types of calcium channels in guinea-pig ventricular myocytes. Proc Nat1 Acad Sci USA 83, 5340-5344. MULLINS LI (1979) The generation of electric currents in cardiac fihres by Na/Ca exchange. Am J Physiol 236, C103-c110. POWELL T, NOBLE D (1989) Calcium movements during each heart beat. Mol Cell Biochem 89, 103-108. PYTKOWSKI B, LEWARTOWSKI B, PROKOPCZUK A, ZDANOWSKI K, LEWANDOWSKA K 11983) Excitationand rcstdependent shifts of Ca in guinea-pig ventricular myocardium. Pfltigers Arch, 398, 103-l 13. RICH TI, LANCER GA, KLASSEN MG (1989). Two components of coupling calcium in single ventricular cells of rabbits and rats. Am J Physiol, 254, H937-H946. SHATTOCK MJ, BERS DM (1989) Rat vs rabbit ventricle: Ca flux and intracellular Na assessed by ion-sclrrtivr microelectrodes. Am J Physiol, 256, C813-C822. STERN MD, SILVERMAN HS, HOUSER SR, JOSEPHSON MC, CAPOGROSSI CG, NICHOLS WJ, LEDERER WJ. LAKATTA EC 1988) Anoxic contractile failure in rat heart myocytes is caused by failure of intracellular calcium release dur trl alteration of action potential. Proc Nat1 Acad Sci USA, 85, 695&6958. WIER WG, BEUCKELMANN DJ (1989) Sodium-calcium exchange in mammalian heart: current-voltage rcalation and intracellular calcium concentration. Mol Cell Biochem, 89, 97-102.
